The long range goal of the proposed work is to define the molecular components and mechanisms mediating Vibrio cholerae colonization and virulence protein secretion to the point where there is sufficient knowledge to intelligently incorporate this information into improved cholera vaccine strategies and antimicrobial therapies designed to inhibit these events. Most of the proposal involves analysis of the molecular mechanisms by which toxin coregulated pilus (TCP) is formed and mediates intestinal colonization. Some steps in the process by which TCP and other type 4 pili are built are linked and/or related to the process of toxin and other virulence determinant secretion by type II secretion systems. Thus further understanding of the mechanisms of type 4 pilus biogenesis should lead to the characterization of potential antimicrobial targets involved in multiple virulence pathways. We will examine the aspects of pilus biogenesis in detail. These experiments will be facilitated by our currently available collections of tcp genetic constructs and immunoreagents. Regarding the mechanism of TCP function, we will utilize the technique of field emission scanning electron microscopy in combination with specifially engineered tcpA missense mutations and a tcpB deletion mutant to address the biophysical mechanisms that lead to pilus supertwist formation, promoting the bacterial associations that represent the basis of microcolony formation in the intestine. Further characterization of TcpF and its role in colonization will be undertaken. This analysis is aided by the recent solution of the crytsal structure of TcpF and the mapping of a functional domain of the protein within this structure. Additional steps in the epithelial interaction with V. cholerae will be defined using carbohydrate binding analyses. Taken together, the results of the proposed studies will provide detailed information regarding the mechanisms of V. cholerae colonization from the levels of defining the components, understanding how they are elaborated to the cell surface and beyond. RELEVANCE (See Instructions): Diarrheal diseases caused by enteric infectious bacteria continue to cause significant morbidity and mortality world-wide, including within the US. Despite years of sophisticated research on V. cholerae, the causative agent of cholera, there is still no effective vaccine. The type 4 pilus, toxin coregulated pilus (TCP), of V. cholerae is the major colonization factor and represents a paradigm for type 4 pilus biogenesis and function throughout the realm of enteric bacteria. The proposed studies are relevant to the prevention and cure of cholera as well as a number of other enteric diseases caused by gram-negative enteric bacterial infections world-wide, which addresses major goals of the NIH mission.